Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor-mediated constitutive and induced expression.
Transcriptional activation of the Cyp1B1 gene in rodents is stimulated by both polycyclic hydrocarbons and cAMP. The mouse Cyp1B1 gene structure contains three exons, of which the second nucleotide of exon 2 is the translation start site. Primer extension analysis identified a transcription start domain defining an exon 1 of 371 base pairs. The sequence 1.075 kilobases upstream of the transcription start site showed 11 xenobiotic-responsive elements (XRE) (TnGCGTG or GCGTG) that are putative aryl hydrocarbon receptor (AhR)-binding sites and three steroidogenic factor-1 motifs that are associated with cAMP-mediated transcriptional activation of genes. A transiently transfected Cyp1B1-luciferase construct, composed of exon 1 and 1.075 kilobases of 5'-flanking region, was induced by 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10.0 +/- 3.0-fold, n = 6) in C3H10T1/2 cells, which exclusively express Cyp1B1. The 90-base pair basal promoter contains two SP-1 sites, one SF-1 site, and a TATA-like box. TCDD induction and basal expression were dependent on positive regulatory elements present between -1075 and -810. Five XRE motifs localized in the enhancer region were completely conserved between mouse and human CYP1B1 sequences. Similar inductions were seen in Hepa-1 cells, which express Cyp1A1 but not Cyp1B1. However, basal Cyp1B1 promoter activities were 4-10-fold higher in C3H10T1/2 cells providing the enhancer region was present, partially reproducing the in vivo cell-specific expression of Cyp1B1. Gel shift experiments established that TCDD stimulates AhR binding to the downstream XRE in the enhancer region. However, oligonucleotides that encompass two other XREs show a high affinity complex of similar size that is evident even without TCDD treatment and that does not contain either the AhR or AhR nuclear translocator. The fourth XRE is immediately adjacent to an E-box, and this oligonucleotide formed a smaller complex that was dependent on this E-box sequence. Negative regulatory sequences have been located between the promoter and TCDD-responsive enhancer regions. Constitutive expression of the Cyp1B1 gene was lost in AhR-deficient cells and was restored by transfected AhR cDNA. Reporter constructs function in a parallel manner, demonstrating the key role of the AhR in constitutive as well as TCDD-induced expression of Cyp1B1 in mouse embryo fibroblasts.
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Department of Pharmacology, Medical Science Center, University of Wisconsin, Madison, Wisconsin 53706, USA.
Savas
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Alexander
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Jefcoate
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology; D004742:Enhancer Elements, Genetic; D004790:Enzyme Induction; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D015698:Genomic Library; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D054339:Steroidogenic Factor 1; D014158:Transcription, Genetic
C515057:NR5A1 protein, human; D009687:Nuclear Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D054339:Steroidogenic Factor 1; C515058:steroidogenic factor 1, mouse; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1